Insulin molecule is composed of two chains – a and P chains of amino acids. Initially it is produced as a single long chain called Pre-proinsulin which is later cut into a shorter chain called proinsulin (composed of 84 amino acid units).
Proinsulin then breaks into a and P chains when a piece from the middle is cut off. Chain a is composed of 21 amino acids and chain p has 30 amino acids – The remaining 33 amino acids act as a connecting peptide chain.
The genes for insulin chain a and P are identified and located. This may be done first by identifying the codons and then the corresponding mRNAs can be reconstructed.
Using these mRNA molecules and with the help of the enzyme reverse transcriptase the corresponding segmentas of double stranded DNA mol ecules are produced. Then with the help of the enzyme terminal transferase, the ends of the insulin genes (DNA strands) are extended with short sequence of identical bases.
Plasmids obtained from E.coli are opened up at specific portions by the use of restriction enzyme.
The insulin genes (DNA strands) of a and p chains are added to the plasmids separately forming recombinant DNA plasmids.
Gaps if any in the circular plasmids of a and p chains are filled by lac operon gene for switching on the synthetic process. The plasmids with recombinant DNA for a and p chains are inserted into E.coli finally.
The recombinant DNA plasmids multiply in E.coli and trigger the synthesis of a and P chains of insulin as a genetic expression. Following large scale production cultures, the insulin chains are obtained following specific purification processes.
Finally the a and P chains are chemically bonded by disulphide bonds to produce the complete molecule of insulin.
Gilbert and Villakomaroff (1980) isolated mRNA for insulin from P cells of the pancrease of rat and inserted it into PBR 322 plasmid in the middle of a gene normally coding for the enzyme penicillinase and incorporated it into the cloning organism (E.coli) E.coli cells produced the hybrid protein consisting of penicillinase + proinsulin. Proinsulin was then separated from penicillinase.